Optimally fluorescent oligonucleotides

ABSTRACT

A method for the preparation of optimally fluorescent oligonucleotides wherein fluorescent dye-conjugated nucleotide triphosphates are incorporated into a nucleic acid sequence in a defined repetitive manner which allows for the optimal specific fluorescence of the oligonucleotide. The oligonucleotides of the present invention are useful in the assay of a wide variety of nucleic acid sequences, specifically wherever highly fluorescent nucleic acid probes are desired.

FIELD OF THE INVENTION

This invention relates to the preparation and use of labeledoligonucleotides. More particularly, it relates to methods of preparingand using fluorescently labeled nucleotides.

BACKGROUND OF THE INVENTION

Many different fluorescent compounds have been used to detect nucleicacids. Broadly, fluorescent labels of nucleic acids can be divided intotwo classes: (1) those which covalently modify nucleic acids with afluorescent moiety, and (2) those which non-covalently modify nucleicacids with a fluorescent moiety, i.e., by ionic interactions,hydrogen-bonding, or intercalation. In general, non-covalent fluorescentprobes of nucleic acids exhibit dramatically increased fluorescence uponbinding to nucleic acids, and consequently, have been very useful inassays designed to determine the total nucleic acid present in a givensample. In addition, non-covalently bound fluorescent molecules can, andwill, migrate from a labeled strand to an unlabeled one. Covalentlybound fluorescent molecules, on the other hand, can not migrate from alabeled oligonucleotide to an unlabeled one. Therefore covalently boundfluorescent moieties are preferred for use as fluorescently taggednucleic acid probes.

Examples of fluorescent compounds which have been covalently attached tonucleic acid sequences include conjugates between nucleotidetriphosphates or phosphoramidites and fluorescent moieties, and directlyreactive dyes. Nucleotide triphosphates are incorporated into a nucleicacids by nucleic acid polymerases. Commercially available nucleotidetriphosphates-dye conjugates include dCTP-Cy3, dCTP-Cy5, dUTP-FluorX,etc. available from DuPont, Molecular Probes, Boehringer Mannheim, andAmersham Life Sciences. These dye conjugates contain cyanine orfluorescein derivatives which are covalently bound to the nucleotide,and each dye conjugate differs with respect to the absorbance maxima ofthe dye moiety. Directly reactive dyes covalently bind to an existingnucleic acid sequence. A few reactive dyes are commercially available,including various psoralens and ethidium mono- and di-azides.

The chemistry associated with conjugates of phosphoramidites andfluorescent molecules has dramatically improved in recent years allowingfor the complete synthesis of fluorescently labeled oligonucleotideswith commercially available nucleic acid synthesizers. Fluorescentlylabeled oligonucleotides have also been synthesized by a combination ofmodified phosphoramidites and reactive dyes, typically involving theincorporation of primary amines in the oligonucleotide during synthesisfollowed by covalent coupling of the amine groups to a reactive dye.

Of the three methods for the covalent linkage of fluorescent compoundsto oligonucleotides, the nucleotide triphosphate-dye conjugates offerthe greatest flexibility and the highest achievable specificfluorescence. Synthetic nucleic acids (molecules producednon-enzymatically) are generally limited to less than 100 bases and aresubject to variable dye coupling chemistries. Directly reactive dyes,such as ethidium monoazide, react non-specifically and can potentiallydamage the labeled oligonucleotide. Polymerase-driven labeling, on theother hand, can produce molecules from a few tens of bases to severalkilobases, can utilize standard labeling methods such as nicktranslation and primer extension reactions, and the degree of dyeincorporation can be roughly controlled by varying the ratio of labeledNTP to unlabeled NTP.

The primary limitation of polymerase-driven fluorescent labeling ofnucleic acids is the absence of absolute control of the amount offluorescent compound incorporated into a particular sequence. Forexample, if one desires to label DNA with dCTP-Cy3 and the specificsequence has only a limited number of "C" sites, then the resultingfluorescently labeled oligonucleotide will have relatively few Cy3molecules and consequently a low specific fluorescence. The presentinvention overcomes this sequence specific limitation and optimizes theincorporation of the fluorescent moiety by polymerase.

SUMMARY THE INVENTION

The invention provides a method for the preparation and purification ofoptimally fluorescent oligonucleotides comprising the steps of (a)preparing a primer; (b) preparing a template oligonucleotide containinga nucleotide sequence complementary to the primer, and a nucleotiderepeat region downstream from the the complementary region; (c)annealing the template and primer in a suitable reaction mediumcontaining a polymerase, nucleotide triphosphates and fluorescentdye-conjugated nucleotide triphosphates; (d) initiating synthesis of acomplementary strand on the template; (e) attaching the oligonucleotidecontaining a target sequence adjacent to the complementary strand; and(f) purifying the optimally fluorescent oligonucleotide by anyappropriate method.

The invention also provides an oligonucleotide comprising a nucleotidesequence complementary to a primer, and a nucleotide repeat regiondownstream from said complementary sequence, wherein the nucleotiderepeat region comprises N^(t) where N^(t) is any nucleotide which canform a base pair with a fluorescent dye-conjugated nucleotidetriphosphate, and optionally, a plurality of nucleotides, N, which arenot capable of forming a base pair with a fluorescent dye-conjugatednucleotide triphosphate.

The invention further provides an optimally fluorescent oligonucleotidecomprising a radiolabeled nucleic acid sequence and a nucleotide repeatregion, wherein the nucleotide repeat region comprises N^(f), whereN^(f) is any nucleotide which is conjugated to a fluorescent dye, andoptimally, a plurality of nucleotides, N, which are not conjugated to afluorescent dye.

These and other embodiments are disclosed or are obvious from thefollowing detailed description.

DETAILED DESCRIPTION OF THE INVENTION

In order to more clearly understand the invention, certain terms aredefined as follows.

The "complement" to a first nucleotide sequence is well known to be asecond sequence comprising those bases which will pair by Watson-Crickhybridization with the first sequence. Thus, the complement to thedeoxyribonucleic acid (DNA) sequence 5'-ATGC 3' is well known to be5'-GCAT 3'. For duplex, or double stranded DNA, each of the two strandsare described as complementary to the other or as a complementary pair.The terms complement and anticomplement may also be used. With referenceto the identification of the strand of duplex DNA from whichtranscription to RNA proceeds, the transcription strand is generallydescribed as plus and its complement as minus (or "+" and "-"), or thetranscription strand may be described as the sense strand, and itscomplement as antisense. Two strands each hybridized to the other havingall base pairs complementary, are 100% complementary to each other. Twostrands, each hybridized to the other, having 5% of basesnon-complementary, are 95% complementary (or the two strands have 95%complementarity).

A "probe" is a single or double stranded nucleic acid which has asequence complementary to a target nucleic acid sequence of interest andwhich has some additional feature enabling the measurement of theprobe-target duplex. The artisan will understand that if the probeand/or the target is double stranded, the double stranded nucleic acidmust undergo strand separation before hybridization can take place.

A probe is rendered detectable by an attached tag or label. A tag orlabel linked to a probe may include, in principle, a fluorescent orluminescent tag, an isotopic label, a dye label, an enzyme label, anantigen determinant detectable by an antibody, or a binding moiety suchas biotin enabling yet another moiety such as a streptavidin coated beadto specifically attach the probe. When the labeled or taggedprobe-target duplex is formed, that duplex may be detected by thecharacteristic properties of the tag or label. The probe with its labelmoiety anneals to the target by hybridization and duplex formationallowing detection by a label.

A "primer" is a relatively short segment of oligonucleotide which iscomplementary to a portion of the sequence of interest (the sequence ofinterest can be a subfragment within a larger nucleic acid sequence). Aprimer represents the 5' terminus of the resulting extension product. Aprimer which is complementary to the sequence of interest on thetemplate strand enables the 3' terminus to be acted on by a polymerase.A primer may also be modified at its 5' end with a binding moiety ordetectable label.

"Hybridization" describes the formation of double stranded or duplexnucleic acid from complementary single stranded nucleic acids.Hybridization may take place between sufficiently complementary singlestranded DNA and/or RNA to form: DNA-DNA, DNA-RNA or RNA-RNA.

The in vitro amplification of DNA is catalyzed by DNA polymerase. Anumber of types of DNA polymerases are known in the art. They generallyshare the common property of catalyzing the synthesis of a doublestranded DNA sequence utilizing a single stranded template to which aprimer is annealed. DNA polymerases extracted from most organisms becomeinactive at the temperatures required for thermal denaturing of nucleicacids. Thus, replacement of the enzyme at the start of each thermalcycle, or the addition of a factor able to prevent heat inactivation, isrequired if such heat sensitive enzymes are utilized. The DNApolymerases which are preferred for in vitro PCR as well as for theinvention are derived from organisms which thrive at high temperaturesand thus are heat resistant, i.e., thus maintain adequate catalyticactivity at the temperature which denatures duplex DNA.

The reaction catalyzed by DNA polymerase is known to the art, andreferred to herein as the "DNA polymerase reaction". The reactionrequires some or all of the four deoxyribonucleotide triphosphates andprimers, preferably in molar excess, and a means for cyclic strandseparation. Strand separation is preferably achieved by thermal cyclingbetween annealing and denaturation temperatures. Reverse transcriptaseis known to mediate both RNA and DNA copying, as well as DNA to DNAcopying. Hence, any number of enzymes now known will catalyze thepolymerization reaction.

"Optimal spacing" describes that distance between fluorescently labelednucleotides which results in the maximum fluorescence of theoligonucleotide.

"Specific fluorescence" refers to the quantum efficiency per unit massof labeled nucleic acid, or the amount of fluorescent label incorporatedper unit mass of labeled nucleic acid.

"Optimal fluorescence" refers to the maximum specific fluorescence whichcan be obtained in a given reaction medium, and it is based on theoptimal spacing of the fluorescent moieties in the oligonucleotide andthe polymerase chosen for a particular fluorescently labeled nucleotide.

"Primer extension" refers to the template directed, polymerase drivenprocess of extending a primer oligonucleotide which is base paired to atemplate with nucleotide triphosphates, such that the final product is a(fully or partially) duplex DNA strand.

A "target sequence" is that oligonucleotide sequence which is to belabeled (either covalently or non-covalently), coupled or ligated to anoptimally fluorescent moiety.

The process of "nick-translation" is catalyzed by DNA polymerase, and itis characterized by the simultaneous polymerization of new DNA and thedegradation of DNA ahead of the growing site.

A "DNA matrix (or matrices)" refers to successive layers ofpolynucleotides of specific structure, including a double-stranded waistand single stranded, free arms at the molecule ends, formed byhybridization of the arms to adjacent molecule arms. Such matrices aredescribed in U.S. Pat. No. Nos. 5,175,270 and 5,487,973, which areincorporated herein by reference.

"Specific activity" refers to that amount of radiolabel present per unitmass of labeled compound, and it is usually expressed in units of Curies(Ci) per millimole (mmol).

The process known as a "Southern blot" enables the detection of specificsequences of a nucleic acid to be detected by a labeled probe. When thelabel is radioactive the result is visualized by autoradiography. Therestricted DNA fragments are denatured in a gel and blotted onto a sheetof membrane nitrocellulose or nylon by capillary action orelectrophoretic transfer in a manner that preserves the originalpattern. After the single-stranded DNA is permanently bound to themembrane, the sheet is incubated in a solution containing labeled probe(i.e., complementary DNA or RNA). Once the homologous sequences have hadtime to anneal, the membrane is washed free of unhybridized probe. Theresulting autoradiograph, for radioactive probes, will indicate whichrestriction fragments bear homology to the nucleotide sequence on theprobe.

Similarly, a "Northern blot" is the analogous process whereby specificsequences of RNA are detected by a labeled probe. The RNA is blottedonto a membrane, and the sheet is incubated in a solution containinglabeled probe. After the complementary sequences have annealed, themedium is washed free of unhybridized probe and the label is detected.The result will indicate which RNA fragments bear homology to thenucleotide sequence on the probe.

A nucleic acid "dot blot" is produced when a nucleic acid in solution isdetected by spotting the solution on a membrane and detected as in aSouthern or Northern blot. Dot blots can be used to quantitate theamount of nucleic acid in an extract.

"Random priming" refers to the process whereby double stranded DNA isdenatured in the presence of random primers, and unlabeled nucleotidetriphosphates, ³² p-labeled nucleotide triphosphates and polymerase areadded to initiate elongation of the primer, followed by denaturation torelease labeled probe.

A "microtitre plate assay" refers to the detection of anantigen-antibody, dye-substrate or probe-target interaction between asolution of unknown concentration of antigen, protein or DNA/RNA. Theunknown solution is placed in a microtitre plate, which consists ofindividual wells for small volumes (usually no more than 200 μl), and isreacted with an antibody solution, dye or probe of known concentration.The degree of interaction between the reactant and unknown solution isindicative of the concentration of the solute present in the unknownsolution. The interaction can be assessed by fluorescence, ultra-violetabsorption, or reaction with a secondary antibody solution.

The method of the present invention generates labeled oligonucleotideswith a known number and spacing of fluorescent moieties in the sequence.The oligonucleotides of the present invention may be represented by theformula:

    N.sup.t (N.sup.t).sub.n N.sup.t

where n is an integer from 20 to 1000; wherein all nucleotides in thesequence are capable of forming a base pair with an optimallyfluorescent dye-conjugated nucleotide triphosphate. Hence, thecorresponding optimally fluorescent oligonucleotide may be representedby the formula:

    N.sup.f (N.sup.f).sub.n N.sup.f

where n is an integer from 20 to 1000; wherein N^(f) represents anoptimally fluorescent nucleotide in the sequence.

Additionally, oligonucleotides of the present invention may berepresented by the formula:

    N.sup.t (N.sub.m N.sup.t).sub.n N.sub.m

where n is an integer from 20 to 1000, and m is an integer from 1 to 11;wherein nucleotide N^(f) is capable of forming a base pair with anoptimally fluorescent dye-conjugated nucleotide triphosphate, andnucleotide, N, is not capable of forming such base pairs.

Hence, the corresponding optimally fluorescent oligonucleotide may berepresented by the formula:

    N.sup.f (N.sub.m N.sup.f).sub.n N.sub.m

where n is an integer from 20 to 1000, and m is an integer from 1 to 11;wherein nucleotide N^(f) represents a fluorescently labeled nucleotidein the sequence, and nucleotide N, is not labeled fluorescently.

The labeling of the target sequence with the fluorescent moiety can bedone prior to or during the incorporation of the target sequence to theoligonucleotide. When the fluorescently labeled oligonucleotide isgenerated prior to the incorporation of the target sequence, the targetsequence can be attached to the fluorescently labeled oligonucleotide byprimer extension or ligation. Alternatively, the fluorescent moietiescan be incorporated with the target sequence during the polymerizationreaction between the target and an appropriate template, with theaddition of dye-conjugated nucleotide triphosphates (NTPs) in additionto unlabeled NTPs, by cloning or randomer extension.

The process begins with the determination of the optimal spacing andpreferred polymerase for each dye-NTP conjugate. In general, a primersequence (preferably 6-40 bases long) and multiple template sequenceswill be required. The template sequences (20-100 bases) will have aprimer binding region and downstream from the primer binding region theappropriate nucleotide ("G" for "C" conjugated dyes, "A" for "U"conjugated dyes, etc.) spaced every base (polyhomonucleotide in a firsttemplate sequence), every other base in a second template sequence,every third base, every forth base, every fifth base, every sixth base,every seventh base, every eight base, every ninth base, every tenth baseor every eleventh base. The repetition of nucleotide in this manner isreferred to herein as a nucleotide repeat region, and it can berepresented by the following formula:

    N.sub.(0-11) N.sup.t

where N represents a nucleotide which is not capable of forming a basepair to a dye-conjugated nucleotide; N^(t) represents the nucleotidewhich is capable of forming a base pair to a dye-conjugated nucleotide,or that which is directly conjugated to the fluorescent dye. The spacingof the dye-conjugated nucleotides within the nucleotide repeat regionshould be as close as possible without quenching the fluorescence signalof the individual moieties. The intervening sequence can be repeatedsequence, semi-repeated, or random sequence selected from the threenon-basepairing (to the dye-NTP) bases. The primary constraint on theintervening sequence is the absence of self homology, eitherintertemplate or intratemplate to minimize non-specific priming events.A single set of primers is sufficient for determining the optimalspacing for any dye-NTP conjugate.

The primer should be radiolabeled, preferably with ³² P, to highspecific activity, and the actual specific activity should be determinedby counting an aliquot of the radiolabeled primer and measuring theoptical density at 260 nm. The actual determination of the specificactivity may be omitted if the optimal spacing is the only informationdesired from the experiment; however, determination of the specificactivity allows for rapid subsequent determination of the specificfluorescence. The 5'-³² P labeled primer and templates (in a separatereaction for each template sequence) should be mixed in approximatelystoichiometric ratios, and allowed to anneal. The annealing process canbe done in any buffer conducive to the formation of nucleic acidhybrids, such as 100 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA. Afterannealing, the sample can be precipitated with ethanol and resuspendedin water, or alternatively used directly in the polymerization assay.

An aliquot, approximately 1 μg, of annealed primer-template should thenbe added to a series of reactions using multiple polymerases, such asSEQUENASET™ from Amersham Life Sciences, Klenow fragment of DNA Poll,Taq Polymerase, Pyrostase, and other commercially available polymerases.The reaction should take place in the optimized buffer for eachparticular polymerase (as determined by the manufacturer). The reactionshould also contain the dye-NTP, and unlabeled NTPs at a concentrationof 20 μM to 2 mM (excluding the NTP which is already added as a part ofthe dye-NTP conjugate). Each polymerase is capable of recognizing andincorporating the dye-NTP conjugates into the polymerization reaction toa different degree, and the choice of enzyme may significantly affectthe specific fluorescence of the labeled probe.

Following the polymerase reaction the labeled oligonucleotides should bepurified away from the unincorporated nucleotide triphosphates. Thepurification can be accomplished by ethanol precipitation, sizeexclusion chromatography, gel electrophoresis or another method. Thepurified labeled oligonucleotides should be quantitated by scintillationcounting or, if sufficiently large quantities are available, bymeasuring the optical density at 260 nm and at the wavelength of maximumabsorbance of the dye moiety.

The specific fluorescence of the purified labeled oligonucleotides isthen determined. A known aliquot of the labeled oligonucleotide isdiluted in reagent grade water and the amount of fluorescence determinedwith a fluorometer, preferably a variable slit spectrofluorometer. Thereaction mixture showing the greatest specific fluorescence is selectedas the optimal labeling method for that particular dye-NTP conjugate.

Alternatively, the methods of the present invention can be used forlabeling ribonucleotide sequences, in which case, RNA polymerase andlabeled ribonucleotides would be used in the synthesis of optimallylabeled oligonucleotides.

Labeling of a Target Sequence

The optimal spacing and the polymerase needed for a particular dye-NTPconjugate are selected as described hereinabove. Subsequently, targetsequences may be labeled by the optimally labeled oligonucleotide byligation of the target sequence to fluorescently labeled nucleotides,cloning the target sequence adjacent to the optimal spacing sequence orby "randomer" extension reaction.

Labeling a Target Sequence by Ligation

Labeling by ligation is accomplished by first synthesizing and purifyingan optimally labeled nucleic acid (20 bases to 2 kilobases). The targetsequence for labeling with the fluorescently labeled oligonucleotide isnicked into small pieces, which average 30-70 bases, by chemicaldegradation or by treatment with nuclease such as DNAse I or arestriction enzyme. Approximately equal weights of fluorescently labeledoligonucleotide (typically 50 ng to 5 μg in 50-100 μl total reactionvolume) and target sequence are reacted in ligation buffer asrecommended by the ligase enzyme manufacturer. The relative success ofthe ligation step can be assessed by gel electrophoresis. The ligatedmaterial can be directly used in hybridization assays or, if desired,purified by precipitation, size fractionation, gel electrophoresis,antigen-specific binding, or another method.

Labeling a Target Sequence by Randomer Extension

The basis of this labeling technique is the use of a short (6-12 base)random sequence at the 3' end of the optimally labeled oligonucleotide.The initial labeling reaction of the template with fluorescent compoundis modified such that the template molecule is designed to have a 5'overhang (the extension region for incorporation of dye-NTP) as well asa 3' overhang of 6-200 bases with the most 3' sequence being a randomsequence of typically 6-12 bases. The purified labeled oligonucleotidemay be used directly in the primer extension reaction or preferablycrosslinked with trimethylpsoralen prior to use in the target labelingreaction.

The target labeling reaction consists of denaturing the desired targetsequence, adding the polymerase, an excess of labeled-primer moleculesand the appropriate NTPs for the desired polynerase (i.e. dATP, TTP,dCTP, dGTP for use with Klenow polymerase), in the appropriate buffer.Some of the 3' ends of the fluorescently labeled randomer will serve asprimers on the target molecule thereby being extended during thepolymerization process and generating molecules having a 3' endcomplementary to the target molecule and an optimally labeled 5' end.

Labeling of a Target Sequence Following Cloning

By cloning the optimized template sequence downstream from the SP6, T3,or T7 promoter sites, and then cloning a target sequence furtherdownstream from the promoter sequence, subsequent polymerase labelingcogenerates polynucleotides having the optimally labeled sequence andthe target sequence. The polymerase may be an RNA polymerase, such as T7RNA polymerase for use with ribonucleotide triphosphates. The polymerasemay also be a DNA polymerase and the labeling performed by specificprimer extension or via random priming methods.

The labeled nucleic acids may be used as probes for a particularsequence wherever highly fluorescent nucleic acid probes are desired,e.g., in known nucleic acid assay methods such as dot blot, Southernblot or Northern blot, etc. In addition, the fluorescently labeledoligonucleotides may be used for in situ hybridization techniques,wherein the sequence of interest is present in only a small number ofcells within a large mixed population. Such sequences may beundetectable in tissue extracts due to the presence of interferingsequences from surrounding tissue.

In situ hybridization may be used to: (1) identify sites of geneexpression; (2) analyze the tissue distribution of transcription; (3)identify and localize viral infection; (4) follow changes in specificmRNA synthesis; and (5) aid in chromosome mapping. The present inventioncan provide increased specific fluorescence and therefore, enhancedsensitivity when compared to conventional methods for in situhybridization.

Another use for the present invention is for the enhanced detection ofnucleic acid sequences in combination with DNA matrices, which aredisclosed in U.S. Pat. Nos. 5,175,270 and 5,487,973, and which areincorporated herein by reference. The DNA matrices disclosed in U.S.Pat. Nos. 5,175,270 and 5,487,973 comprise successive layers ofpolynucleotides having both single and double-stranded regions. Theoligonucleotide probes of the present invention can be hybridized to thenon-annealed, free, single-stranded arms of the DNA matrices, and theresulting fluorescently labeled DNA matrices can be useful in the assayof a wide variety of nucleic acid sequences including those associatedwith pathogenic bacteria and viruses.

Finally, the present invention can be used in a microtitre plate assaysystem based on fluorescence, wherein the high specific fluorescenceprovided by the optimally fluorescent oligonucleotide probes wouldenhance and facilitate the detection of the fluorescent moiety in theassay.

EXAMPLE 1

Fluoresoence Optimization of the Incorporation of dCTP-Cy3

In this example the polymerase and optimal spacing was determined fordCTP-Cy3 incorporation. In addition, it was desirable that thefluorescently labeled strand be longer than the template strand so theprimer sequence had a 5' overhang relative to the template strand. Thetemplate therefore utilized only three bases, "G", "A", and "T", so that"back" reaction (extension of the template sequence on the primersequence) could be blocked by omitting dGTP from the reaction buffer. Bydesigning the reaction to allow a 5' overhang on the primer strand,subsequent strand separation could be readily achieved by denaturing gelelectrophoresis, since in the post reaction, the extension product islonger than the template sequence.

The templates were 41 mers designated a(+)-2C, a(+)-3C and a(+)-4C, eachdesigned to incorporate the dCTP-Cy3 dye every other, every third, orevery fourth base respectively. The primer sequence was a 31 merdesigned to hybridize with the template strand over 14 bases. Fullextension of the primer was expected to yield a 58 mer, with 27 basesadded by the polymerase reaction. ##STR1##

The synthetic oligonucleotides were purchased from The Midland CertifiedReagent Co., Midland, Tex. and were dissolved in reagent grade water ata concentration of 200 ng/μl (based on 30 μg/ml=1 A260U). The primersequence was 5' labeled with ³² P by γ³² P-ATP (ICN Radiochemicals Cat #35020) 100 uCi/ reaction and 10U of polynucleotide kinase (BoehringerMannheim Biochemicals) in the manufacturer's supplied reaction bufferand recommended reaction time. The primer was purified essentially freeof unincorporated nucleotide by size exclusion chromatography(select-D-G25 Column 5'-3'®, Boulder, Colo.) as recommended by themanufacturer, and it had a specific activity of 31,180 cpm/ng. Theprimer was stored in aliquots, each at a concentration of 62.2 ng/μl (asdetermined by the OD₂₆₀ of 58.3 μl in 1 ml of reagent grade water) in100 mM Tris-HCl, pH 8.0, containing 200 mM NaCl and 1 mM EDTA.

The annealing reaction was carried out by reacting 25 μl templateoligonucleotide (5 μg, 0 cpm) and 49 μl³² P labeled primer (3.0 μg,39,540,000 cpm) in 24 μl reagent grade water containing 2.0 μl 5M NaCl(final concentration of 100 mM NaCl). The reaction was cooled from 95°C. to room temperature over 15 minutes in a 1L beaker.

The subsequent polymerase extension reaction was carried out bycombining 10.0 μl of the aforementioned annealed oligonucleotidereaction mixture, 10 μl 5X reaction buffer (supplied by the manufacturerof the polymerase), 1 μdATP, 1 μl dTTP (each 10 mM, supplied byBoehringer Mannheim), 5.0 μl dCTP-Cy3 (1 mM, supplied by BiologicalDetection Systems), 22 μl reagent grade water and 1 μl SEQUENASE™ (USBUnited States Biologicals, 10 units) or Klenow fragment of DNA Poll(supplied by Boehringer Mannheim, 10 units). The reaction was completeafter 1 hour at room temperature.

A portion of each reaction was loaded on a 9% denaturing polyacrylamidegel. Following electrophoresis, the gel was dried on 3 MM paper andexposed to x-ray film for autoradiography. Then, a separate aliquot fromeach of the reactions was loaded on a preparative 9% denaturingacrylamide gel, electrophoresed, and stained with ethidium bromide. Thelabeled (5'³² P and 3' Cy3-CTP at varying spacing) 58 mers were excisedfrom the gel, triturated with 200 μl 10 mM Tris-HCl, pH 8.0, containing1 mN EDTA, and the samples were shaken overnight in 1.51 mlmicrocentrifuge tubes at 37° C. The samples were briefly centrifuged andthe supernatant was transferred to a fresh microcentrifuge tube. Analiquot of each supernatant was counted in a Beckman LS8100scintillation counter. Equal counts (10,000 cpm=854 pg of DNA as 30 mera(-)) were added to 2 ml of reagent grade water and scanned forfluorescence with a SPEX instruments Fluoromax spectrofluorometer.

Excitation was found to have maximum signal to noise ratio at 535 nm.Emission was determined over the range of 560 nm to 620 nm. Emissionmaxima were centered around 565 nm and the emission maximum for eachreaction was determined for the calculation of the specificfluorescence. The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Specific Fluorescence as a Function of Polymerase                                         Maximum Fluorescence                                                                         Specific Fluorescence                              Sample      cps            cps/pg                                             ______________________________________                                        Klenow Rxn 2C                                                                               125,000        146                                              Klenow Rxn 3C                                                                               99,000         116                                              Klenow Rxn 4C                                                                               160,000        187                                              Sequenase Rxn 3C                                                                          1,367,000      1,600                                              Sequenase Rxn 4C                                                                          1,020,000      1,200                                              ______________________________________                                         where cps = counts per second; and pg = picogram                         

The above analysis demonstrates that the choice of enzyme can have asignificant effect on the resulting specific fluorescence, and thespacing of the dye incorporation is important for maximizing thespecific fluorescence. For the dye-nucleotide conjugate, dCTP-Cy3, theoptimal enzyme is SEQUENASE® and the optimal spacing is every thirdmolecule.

EXAMPLE 2

Incorporation of Optimally Fluorescent Oligonucleotide with DNA Matriz

The 3C optimally fluorescent oligonucleotide can be used to label theouter layer of polynucleotides of a DNA matrix, through theirnon-annealed, free, single-stranded arms. The fluorescently labeled DNAmatrix can be used to recognize the multiple DNA arms of the sequencebound to a smaller bead, and to supply an easily measured mass to theassay system.

First, the DNA bead matrix is assembled as described in U.S. Pat. No.5,487,973. Sequential additions of matrix monomers leads to a DNA matrixwith k layers (k-Mmer). The double-stranded, unpurified 3C optimallyfluorescent oligonucleotide (which has a 5' single-stranded overhang) isadded as the final addition to the k-Mmer, yielding a DNA bead matrixhaving optimally fluorescent single-stranded arms. The annealingreaction is performed in 2×SSPE (20×SSPE=3.6M NaCl, 0.2M sodiumphosphate, pH 7.0, and 0.02M EDTA).

Having thus described in detail certain preferred embodiments of thepresent invention, it is to be understood that the invention defined bythe appended claims is not to be limited by particular details set forthin the above description, as many apparent variations thereof arepossible without departing from the spirit or scope thereof.

What is claimed is:
 1. An oligonucleotide comprising a nucleotide repeatregion having the formula:

    (N.sub.m N.sup.f)

wherein N represents any nucleotide which is not conjugated to afluorescent dye moiety, N^(f) represents a nucleotide conjugated to afluorescent dye moiety, and m is an integer from 1 to 11, and whereinfor each said repeat N^(f) and the fluorescent moiety are the same, andfurther wherein m and said fluorescent dye moiety are selected such thatsaid oligonucleotide exhibits maximum specific fluorescence.
 2. Theoligonucleotide of claim 1, further comprising a radiolabled nucleotidesequence.
 3. The oligonucleotide of claim 2, wherein said radiolabel is³² P.
 4. The oligonucleotide of claim 1, which is single-stranded. 5.The oligonucleotide of claim 1, which is double-stranded.
 6. Theoligonucleotide of claim 1, which contains from about 20 to about 1000total nucleotides.
 7. The oligonucleotide of claim 1, linked to a probethat hybridizes with a nucleic acid of interest.
 8. The oligonucleotideof claim 1, in combination with a DNA matrix.
 9. An oligonucleotidecontaining a single fluorescent dye moiety and prepared by a methodcomprising:(a) preparing a primer; (b) preparing a templateoligonucleotide, said template oligonucleotide containing a nucleotidesequence complementary to said primer, and a nucleotide repeat regiondownstream from said complementary nucleotide sequence, said nucleotiderepeat region having the formula:

    (N.sub.m N.sup.t)

wherein m is an integer from 1 to 11, N is a nucleotide which cannotform a base pair with a fluorescent dye-conjugated nucleotidetriphosphate, and N^(t) is a nucleotide which can form a base pair withthe fluorescent dye-conjugated nucleotide triphosphate, wherein for eachrepeat, N^(t) is the same; (c) annealing the template and the primer ina reaction medium comprising a polymerase, unlabeled nucleotidetriphosphates and a predetermined concentration of said fluorescentdye-conjugated nucleotide triphosphate, wherein said polymerase and mare chosen to cause said optimally fluorescent oligonucleotide toexhibit maximum specific fluorescence; (d) initiating synthesis of acomplementary strand on the template which defines said optimallyfluorescent oligonucleotide; and (e) isolating said oligonucleotide fromthe reaction medium, wherein m, said fluorescent dye moiety, and saidpolymerase are selected such that said oligonucleotide exhibits maximumspecific fluorescence.
 10. The oligonucleotide of claim 9, wherein saidmethod further comprises step (f) attaching said oligonucleotide to aprobe that hybridizes with a nucleic acid of interest.
 11. Theoligonucleotide of claim 10, wherein said attaching comprises ligation.12. The oligonucleotide of claim 10, wherein said attaching comprisesrandomer extension.
 13. The oligonucleotide of claim 10, wherein saidattaching comprises cloning.
 14. The oligonucleotide of claim 9, whereinsaid (e) isolating comprises precipitation, size fractionation, gelelectrophoresis or antigen-specific binding.